The development of a fluorescence/colorimetric biosensor based on the cleavage activity of CRISPR-Cas12a for the detection of non-nucleic acid targets.

Nano-biosensors are of great significance for the analysis and detection of important biological targets. Surprisingly, the CRISPR-Cas12a system not only provides us with excellent gene editing capabilities, it also plays an important role in biosensing due to its high base resolution and high levels of sensitivity. However, most CRISPR-Cas12a-based sensors are limited by their recognition and output modes, are therefore only utilized for the detection of nucleic acids using fluorescence as an output signal. In the present study, we further explored the potential application of CRISPR-Cas12a and developed a CRISPR-Cas12a-based fluorescence/colorimetric biosensor (UCNPs-Cas12a/hydrogel-MOF-Cas12a) that provides an efficient targeting system for small molecules and protein targets. These two sensors yield multiple types of signal outputs by converting the target molecule into a deoxyribonucleic acid (DNA) signal input system using aptamers, amplifying the DNA signal by catalyzed hairpin assembly (CHA), and then combining CRISPR-Cas12a with various nanomaterials. UCNPs-Cas12a/hydrogel-MOF-Cas12a exhibited prominent sensitivity and stability for the detection of estradiol (E2) and prostate-specific antigen (PSA), and was successfully applied for the detection of these targets in milk and serum samples. A major advantage of the hydrogel-MOF-Cas12a system is that the signal output can be observed directly. When combined with aptamers and nanomaterials, CRISPR-Cas12a can be used to target multiple targets, with a diverse array of signal outputs. Our findings create a foundation for the development of CRISPR-Cas12a-based technologies for application in the fields of food safety, environmental monitoring, and clinical diagnosis.